The efficiency of quartz addition on electric arc furnace (EAF) carbon steel slag stability.

Electric arc furnace slag (EAF) has the potential to be re-utilized as an alternative to stone material, however, only if it remains chemically stable on contact with water. The presence of hydraulic phases such as larnite (2CaO SiO2) could cause dangerous elements to be released into the environment, i.e. Ba, V, Cr. Chemical treatment appears to be the only way to guarantee a completely stable structure, especially for long-term applications. This study presents the efficiency of silica addition during the deslagging period. Microstructural characterization of modified slag was performed by SEM and XRD analysis. Elution tests were performed according to the EN 12457-2 standard, with the addition of silica and without, and the obtained results were compared. These results demonstrate the efficiency of the inertization process: the added silica induces the formation of gehlenite, which, even in caustic environments, does not exhibit hydraulic behaviour.

Clinical and molecular analysis of haemoglobin H disease in Sardinia: haematological, obstetric and cardiac aspects in patients with different genotypes.

In this study, 251 Sardinian patients (187 adults and 64 children) with haemoglobin (Hb) H disease were evaluated. Two-hundred and sixteen patients (86%) had the deletional type (- -/-alpha) and 36 (14%) patients had the non-deletional type (- -/alpha(ND)alpha). A clear genotype-phenotype correlation was found, with the non-deletional type more severe than the deletional type. Diagnosis of Hb H disease was incidental in about 60% of cases. Aplastic crises due to B19 parvovirus infection were found in five patients (2.1%), while 23 patients (9.6%) experienced one or more haemolytic crises. Nineteen patients with Hb H received sporadic red blood cell transfusions and three patients were repeatedly transfused. Forty-seven of 61 married women (77%) had 82 pregnancies. In children, mean serum ferritin was 87 +/-92 mug/l and in adults, was 192 +/- 180 mug/l in females and 363 +/- 303 mug/l in males. For the 98 male patients, a significant correlation was found between ferritin values and age (r2 = 0.33, P < 0.0001). In the Sardinian population, Hb H disease needs regular monitoring for early detection and treatment of possible complications, such as worsening of anaemia that may require red cell transfusion, cholelithiasis and iron overload.

Cholelithiasis and Gilbert's syndrome in homozygous beta-thalassaemia.

Cholelithiasis has been reported with a variable incidence in homozygous beta-thalassaemia, the reasons for which have only partially been defined. Disease-associated factors or specific modifier genes may be implicated. We assessed the prevalence of cholelithiasis and the effect of co-inherited Gilbert's syndrome genotype on its development in 261 thalassaemia major (TM) and 35 thalassaemia intermedia (TI) patients. Cholelithiasis was found in 20.3% of TM and in 57.1% of TI patients. Its incidence was higher (P < 0.05) in patients homozygous for the (TA7) motif in the promoter of the UGT1-A1 gene, the genotype associated with Gilbert's syndrome, which seems to be a risk factor for the development of gallstones in TM and TI patients.

Hyperbilirubinemia, glucose-6-phosphate-dehydrogenase deficiency and Gilbert's syndrome.

The pathogenesis of neonatal hyperbilirubinemia has not yet been completely defined in normal and glucose-6-phosphate-dehydrogenase (G6PD)-deficient newborns. The recent identification of a variant promoter in the gene encoding for the bilirubin uridine-diphosphoglucuronosyl-transferase (UGT-1 A) associated with Gilbert's syndrome, allowed us to explore whether the presence of this variant promoter is a risk factor for the development of neonatal hyperbilirubinemia in normal newborns and in association with G6PD deficiency. We found that the variant (TA)7/(TA)7 promoter shows no statistically significant difference in normal or G6PD-deficient newborns developing severe hyperbilirubinemia and in control subjects from the same population. This finding indicates that the variant promoter of UGT-1 A does not contribute to the development of hyperbilirubinemia in the newborn.

Soluble transferrin receptor as a potential determinant of iron loading in congenital anaemias due to ineffective erythropoiesis.

Congenital anaemias due to ineffective erythropoiesis may be associated with excessive iron absorption and progressive iron loading. We investigated whether the soluble transferrin receptor (TfR) level was related to the degree of iron overload in 20 patients with thalassaemia intermedia, six patients with congenital dyserythropoietic anaemia type II (CDA II) and four patients with X-linked congenital sideroblastic anaemia (XLSA). All but two patients had increased serum ferritin levels (median 601 microgram/l, range 105-2855 microgram/l). Multiple regression analysis showed that 62% (P < 0.0001) of the variation in serum ferritin was explained by age and by changes in soluble TfR.

Temporal and spatial regulation of symplastic trafficking during development in Arabidopsis thaliana apices.

Plasmodesmata provide symplastic continuity linking individual plant cells. However, specialized cells may be isolated, either by the absence of plasmodesmata or by down regulation of the cytoplasmic flux through these channels, resulting in the formation of symplastic domains. Maintenance of these domains may be essential for the co-ordination of growth and development. While cells in the center of the meristem divide slowly and remain undifferentiated, cells on the meristem periphery divide more frequently and respond to signals determining organ fate. Such symplastic domains were visualized within shoot apices of Arabidopsis, by monitoring fluorescent symplastic tracers (HPTS: 8-hydroxypyrene 1,3,6 trisulfonic acid and CF: carboxy fluorescein). Tracers were loaded through cut leaves and distributed throughout the whole plant. Confocal laser scanning microscopy on living Arabidopsis plants indicates that HPTS moves via the vascular tissue from leaves to the apex where the tracer exits the phloem and moves symplastically into surrounding cells. The distribution of HPTS was monitored in vegetative apices, and just prior to, during, and after the switch to production of flowers. The apices of vegetative plants loaded with HPTS had detectable amounts of tracer in the tunica layer of the meristem and in very young primordia, whereas the corpus of the meristem excluded tracer uptake. Fluorescence signal intensity decreased prior to the onset of flowering. Moreover, at approximately the time the plants were committed to flowering, HPTS was undetectable in the inflorescence meristem or young primordia. Later in development, after several secondary inflorescences and mature siliques appeared, inflorescence apices again showed tracer loading at levels comparable to that of vegetative apices. Thus, analysis of fluorescent tracer movement via plasmodesmata reveals there is distinct temporal and spatial regulation of symplastic domains at the apex, dependent on the developmental stage of the plant.

Alpha-thalassemia carrier identification by DNA analysis in the screening for thalassemia.

Differentiation between heterozygous alpha-thalassemia and several phenotypically resembling alleles at the beta-globin gene cluster such as coinherited delta- and beta-thalassemia or gammadelta beta-thalassemia is a critical step in genetic counseling. In this paper we report our experience in the identification of the alpha-thalassemia carrier state using polymerase chain reaction (PCR)-based methods, and the feasibility and simplification of screening for thalassemia using this approach. Alpha-globin genotype was determined by PCR-based method in 526 adult subjects with reduced mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH), normal hemoglobin A2 and F, and normal serum iron. To verify the reliability of the protocol used, in 68 of these subjects we performed globin chain synthesis analysis and in 101 we determined alpha-globin genotype by Southern blot analysis. Five hundred twenty-one (99%) of 526 subjects examined were identified as carriers of one or two alpha-thalassemia alleles. The identification of the alpha-thalassemia carrier state may be fast and accurate by PCR-based method, avoiding other cumbersome and expensive methods such as globin chain synthesis and Southern blot analysis.

Hyperbilirubinaemia in heterozygous beta-thalassaemia is related to co-inherited Gilbert's syndrome.

The reasons why heterozygotes for beta-thalassaemia have considerable variation in serum bilirubin levels are unknown. High levels of bilirubin could be related to the co-inherited Gilbert's syndrome, determined either by mutations of the coding region or by variation in the A(TA)nTAA motif of the promoter of the bilirubin UDP-glucuronosyltransferase gene (UGT-1). We sequenced the coding and the promoter region of UGT-1A or characterized the A(TA)nTAA motif of the promoter by denaturing gel electrophoresis of radioactive amplified products. The results were correlated with bilirubin levels in 49 beta-thalassaemia heterozygotes for codon 39 (CAG --> TAG) nonsense mutation. 21 normal individuals and 32 unrelated patients with Gilbert's syndrome served as controls. The coding sequence region of the UGT-1A was normal. Five beta-thalassaemia heterozygotes, who were homozygous for the extra (TA) bases in the A(TA)nTAA element of the promoter of UGT-1A, the configuration present in homozygosity in Gilbert's syndrome, had higher bilirubin levels compared to those with the (TA)6/(TA)7 or (TA)6/(TA)6 configurations. In the group of 32 patients with Gilbert's syndrome, 31 of whom had the (TA)7/(TA)7 configuration, we detected 14 heterozygotes for beta-thalassaemia, a figure much higher than predicted on the basis of the carrier rate. Homozygosity for the (TA)7 motif, the typical promoter configuration of Gilbert's syndrome, is one of the factors determining hyperbilirubinaemia in heterozygous beta-thalassaemia.

Hb Puttelange [beta 140(H19)Ala-->Val] in an Italian man with polycythemia.

Hb Puttelange [beta 140(H18)Ala-->Val] was found in a 51-year-old Italian man who had mild polycythemia. The variant eluted from ion exchange high performance liquid chromatography at a position between Hb A and Hb A2. It comprised approximately 34% of the total hemoglobin, was weakly unstable and exhibited an increased oxygen affinity. Amplification of the beta-globin exons and nucleotide sequencing revealed a heterozygosity for a GCC-->GTC mutation in codon 140 corresponding to an Ala-->Val replacement. This substitution accounts for the altered functional properties, probably by producing indirect perturbation of the 2 3-diphosphoglycerate pocket through the nearby lysine residue at beta 82(EF6).

Simultaneous automated determination of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities in whole blood.

We report a potentiometric fully automated method for determining red cell glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase activities and the glucose 6-phosphate dehydrogenase/6-phosphogluconate dehydrogenase index using 25 microliters of whole blood. No sample pre-treatment (e.g., preparation of the haemolysate) is needed and the measurements are performed at pH 8.0 and 37 degrees C under the conditions recommended by the ICSH committee. The reproducibility was constantly good, with within-run CV of 1.0% (glucose 6-phosphate dehydrogenase) and 5.9% (glucose 6-phosphate dehydrogenase/6-phosphogluconate dehydrogenase) for activities in glucose 6-phosphate dehydrogenase non-deficient adults, and of 2.3% (glucose 6-phosphate dehydrogenase, G6PD) and 2.5% (glucose 6-phosphate dehydrogenase/6-phosphogluconate dehydrogenase) for G6PDMediterranean heterozygotes. Linearity was observed up to an activity of 2800 U/l of glucose 6-phosphate dehydrogenase. Results of glucose 6-phosphate dehydrogenase activity (U/l) in whole blood (y) correlated well with those obtained with the previously described monostarter assay, performed at pH 9.2 (y = 0.60x + 37; n = 80; r = 0.991). Results of 6-phosphogluconate dehydrogenase (U/l) in whole blood (y) correlated well with those obtained by the ICSH recommended method (x) (y = 0.779x - 44; n = 23; r = 0.991). Reference intervals are reported for glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and glucose 6-phosphate dehydrogenase/6-phosphogluconate dehydrogenase index relatively to normal, beta- and alpha-thalassaemic glucose 6-phosphate dehydrogenase non-deficient adults, to glucose 6-phosphate dehydrogenase deficient adult males and to G6PDMediterranean non-thalassaemic heterozygotes. We demonstrate that the diagnostic sensitivity of the glucose 6-phosphate dehydrogenase/6-phosphogluconate dehydrogenase index in detecting the G6PDMediterranean heterozygotes is superior to that of the glucose 6-phosphate dehydrogenase activity alone.

Relationship between transfusion regimen and suppression of erythropoiesis in beta-thalassaemia major.

In the management of beta-thalassaemia major, different transfusion schemes are employed with baseline haemoglobin levels ranging from 8 to over 12 g/dl. We studied the relationship between transfusion regimen and suppression of erythropoiesis in 52 patients with beta-thalassaemia major whose mean pretransfusion haemoglobin levels ranged from 8.6 to 10.9 g/dl. Multiple, regression analysis showed that serum transferrin receptor was the parameter more closely related to mean pretransfusion haemoglobin (r = -0.77, P < 0.001). As measured through serum transferrin receptor, erythroid activity was 1-2 times normal for pretransfusion haemoglobin levels between 10 and 11 g/dl. 1-4 times normal for levels from 9 to 10 g/dl, and 2-6 times normal for levels from 8.6 to 9 g/dl. Mean pretransfusion haemoglobin was also inversely related to serum erythropoietin (r = -0.72, P < 0.001), whereas it showed no or a weak relationship with Hb F, reticulocyte count, or circulating nucleated red cell count. This study suggests that serum transferrin receptor is a reliable indicator of suppression of erythropoiesis in beta-thalassaemia major. On the basis of our findings, pretransfusion haemoglobin values of < or = 9 g/dl should be adopted with caution, because these levels can be associated with an insufficient inhibition of erythroid marrow expansion. However, a transfusion programme, with a baseline haemoglobin of 9-10 g/dl, may provide enough suppression of erythropoiesis and allow a reduction in blood consumption as compared with the classic hyper- or supertransfusion schemes. Since fixed haemoglobin levels may not be the best target for transfusion treatment in all thalassaemic patients, assay of serum transferrin receptor may be helpful for individualizing the transfusion regimens.

Evaluation of an automatic HPLC analyser for thalassemia and haemoglobin variants screening.

In this paper the authors report the evolution of a new automatic HPLC analyser for screening haemoglobinopathies. HbA(2) and F determinations are accurate and reproducible. The analysis time is short (6.5 min) and there is a good separation between the HbA(2) values of beta-thalassemia carriers from normals and alpha-thalassemia carriers, with no overlap between these groups. In addition, the system is also able to detect and quantitate most of the haemoglobin variants, particularly those (HbS, HbC, HbE and Hb Lepore) able to interact with beta-thalassemia and could make haemoglobin electrophoresis unnecessary in all samples. The ease of operation and the limited technical work make this system especially suitable for laboratories with a high workload and allow the cost of screening to be reduced.

Genotype of subjects with borderline hemoglobin A2 levels: implication for beta-thalassemia carrier screening.

In this study, we have defined by molecular analysis, the alpha, beta, and delta globin genotype in a group of individuals with normal or thal-like red cell indices but borderline hemoglobin (Hb)A2 levels, who were identified in a program for beta-thal carrier screening. In 37 of 125 individuals with borderline HbA2 levels, we detected a molecular defect in the beta, in both the delta and the beta, or in the alpha globin gene. Specifically seven of these subjects were carriers of the -101 C T mutation, ten of the IVSI nt6 T C mutation, 16 were double heterozygotes for delta and beta thal, and two had the triple alpha globin gene and two the single alpha globin gene deletion. From these results, we may conclude that subjects with borderline HbA2, particularly when they marry a typical beta-thal carrier, should be extensively investigated in order not to miss heterozygous beta-thalassemia.

Serum erythropoietin and erythropoiesis in high- and low-fetal hemoglobin beta-thalassemia intermedia patients.

Clinical data suggest that in beta-thalassemia-intermedia patients, higher levels of circulating fetal hemoglobin (HbF) are associated with greater disease severity at comparable degrees of anemia. We assessed the influence of the amount of circulating HbF on serum erythropoietin (s-Epo) levels and on serum transferrin receptor, a measure of erythropoiesis, in 30 beta-thalassemia-intermedia patients. Twenty-four showed more than 40% HbF (21 of whom with beta (0)-thalassemia) and 6 presented lower HbF levels (beta(+)-thalassemia). The two groups of patients did not differ in age (15.3 v 19 years, respectively) or degree of anemia (Hb = 8.8 g/dL in both groups). Log (s-Epo) was correlated inversely with Hb (r = -0.47; P < .01), and directly with HbF (r = .55; P < .001). Multivariate regression analysis showed that Hb and HbF were independently correlated with s-Epo levels. High-HbF patients had greater s-Epo values at the same Hb level than low-HbF patients. Considering that iron-deficiency anemia control patients represented the predicted physiologic response of s-Epo to anemia, the observed/predicted s-Epo ratio in low-HbF thalassemic patients was no different from controls, but was increased in the high-HbF group. High-HbF patients also showed an expansion of erythropoiesis as much as four to nine times the normal value at the same Hb level as low-HbF patients. We conclude that HbF exerts an independent regulatory effect on erythropoietin production and erythropoiesis that is detectable only when HbF levels exceed 40%.

Normal individuals with high Hb A2 levels.

Increased haemoglobin (Hb) A2 levels associated with reduced mean corpuscular volume (MCV) and Hb content per cell (MCH) are the most typical features of heterozygous beta thalassaemia. However, double heterozygotes for alpha and beta thalassaemia may have normal MCV and MCH but Hb A2 always in the carrier range. In this report we describe two Sardinian families who have increased Hb A2 levels, normal red blood cell indices and normal globin chain synthesis and in whom DNA sequence analysis of beta and delta globin genes did not reveal any abnormality. Our findings demonstrate the existence of a genetic trait not resulting from a defect of the beta globin gene cluster, transmitted in a dominant manner and manifested as isolated increase of Hb A2.

HbH disease in Sardinia: molecular, hematological and clinical aspects.

In this study we have defined the molecular basis and correlated the clinical phenotype with the alpha-globin genotype in a large series of patients of Sardinian descent with HbH disease. The most prevalent molecular defect was the deletion of 3 alpha-globin structural genes most commonly the (--/-alpha 3.7) genotype (83.6%) and rarely the (--/-alpha 4.2) genotype (1.4%), followed in decreasing order of incidence by the combination of deletion alpha zero-thalassemia and initiation codon mutation of the alpha 2-gene (--/alpha NcoI alpha = 9.8%), deletion alpha zero-thalassemia and pentanucleotide deletion of IVS-I of the alpha 2-globin gene, (--/alpha HphI alpha = 3.3%) deletion alpha zero-thalassemia and initiation codon mutation of the alpha 1-gene (--/alpha alpha NcoI = 1.3%), a homozygous state for initiation codon mutation of the alpha 2-gene (alpha Nco alpha/alpha NcoI alpha = 0.7%). Patients with the (--/alpha thal alpha) genotypes showed severer clinical and hematological features as compared to those with the (--/-alpha) or those with the (--/alpha alpha thal) genotypes. The single patient with the (alpha Nco alpha/alpha Nco alpha) genotype had a clinical phenotype intermediate between HbH disease and the alpha-thalassemia carrier status. This heterogeneity depends on the fact that the alpha 2-globin gene produces 2-3 times alpha-globin chains than the alpha 1-gene and the single remaining alpha 1-like globin gene in the -alpha 3.7 chromosome has a compensatory increase in the alpha-globin chain output. alpha-Globin gene mapping of HbH disease patients may be useful for predicting the clinical outcome and to improve genetic counseling.

Iron stores and iron deficiency anemia in children heterozygous for beta-thalassemia.

This paper describes the status of iron stores, the incidence and the hematological characteristics of iron deficiency anemia in children heterozygous for beta-thalassemia. In beta-thalassemia heterozygotes, iron stores were similar to the controls in infancy and tended to increase with age, reaching levels of moderate iron overload solely in adult males. Iron deficiency anemia occurred less frequently in children heterozygous for beta-thalassemia as compared to normal controls, while no difference between the two groups was observed in the incidence of iron deficiency. Ineffective erythropoiesis, typical of heterozygous beta-thalassemia, by causing an increase of iron absorption may limit the effect of iron shortage. At similar levels of iron depletion, however, children heterozygous for beta-thalassemia develop a more severe anemia as compared to non beta-thalassemic children. With the exception of two children, HbA2 levels in the presence of iron deficiency anemia remain in the range of heterozygous beta-thalassemia. In conclusion, our results indicate that children heterozygous for beta-thalassemia have normal iron stores but are relatively protected against the development of iron deficiency. When iron deficiency anemia develops, its clinical expression is usually more severe than in non beta-thalassemic children.